Compositions of thermostable nucleic acid polymerases are useful for amplification of nucleic acids by multiple cycles of the polymerase chain reaction. Various compositions for stabilizing polymerases using surfactants have been disclosed. In an early study it was observed that viral DNA polymerase activity was stimulated and stabilized against thermal inactivation by nonionic detergent (see, Wu and Cetta, Biochemistry (1975) 14(4):789-795). U.S. Pat. No. 6,127,155 discloses stabilization of thermostable DNA polymerases in a composition containing non-ionic polymeric detergents. U.S. Pat. No. 6,242,235 discloses cationic polyethoxylated amine surfactants as polymerase stabilization agents, WO 2008152102 (Liu et al.) and US Pat. Pub. No. 20100099150 (Fang et al.) disclose polymerase stabilization by anionic detergents. US Pat. Pub. No. 20080064071 (Hogrefe et al.) discloses zwitterionic detergents for storage and use of DNA polymerases and US Pat. Pub. No. 2008145910 (Ward et al.) discloses stabilization of DNA polymerase using anionic or zwitterionic detergents during thermal cycling.
Amplification of nucleic acids involves the thermal cycling of a reaction mixture containing a nucleic acid polymerase to generate an amplified target nucleic acid. An example of this thermal cycling process is that which occurs in Polymerase Chain Reaction (PCR), a laboratory technique that can theoretically take one molecule of DNA and produce measurable amounts of identical DNA in a short period of time. PCR is a widely used method in the fields of biotechnology, forensics, medicine, and genetic research. In this method, oligonucleotides are used as primers for a series of synthetic reactions that are catalyzed by a DNA polymerase. The reaction mixture is subjected to multiple cycles of denaturation, annealing, and synthesis performed at different temperatures. Thermostable polymerases are generally used to amplify the target nucleic acid sequences in these thermal cycling reactions because they are not inactivated by the heat denaturation step and, therefore, do not need to be replaced in every round of the amplification cycle. Although efficient, exponential amplification of target sequences is not an unlimited process. Under normal reaction conditions, the amount of DNA polymerase becomes limiting after a certain number of cycles of amplification.
Attempts have been made to improve the PCR amplification process by employing detergents and/or surfactants. For example, U.S. Pat. No. 6,127,155 discloses that the non-ionic detergents NP-40 and Tween stabilize Taq DNA polymerase. However, this patent does not disclose the use of non-detergent surfactants or zwitterionic detergents for the stability of thermostable polymerases in PCR reactions. As another example, US Pat. Pub. No. 20030017567 discloses a method for performing an amplification reaction utilizing a dye that converts electromagnetic energy into thermal energy to heat the reaction mixture. A zwitterionic surfactant is added to the reaction mixture to reduce interference of the dye with the functioning of the nucleic acid polymerase. Additionally, U.S. Pat. Pub. No. 20020168658 discloses the use of zwitterions in combination with a compound that disrupts base pairing, e.g., DMSO, to improve the amplification of nucleic acids that are G+C rich. However, this publication does not disclose the use of zwitterionic detergents alone in improving the amplification of nucleic acids and actually teaches that the zwitterionic detergents used should be selected carefully so as not to inhibit the activity of the DNA polymerase in the reaction.
Given the widespread use and importance of thermal cycling processes, there is a need in the art for ways to improve the stability and/or enhance the activity of thermostable enzymes used in DNA amplification.